A Pilot Study on Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates From Nepal

DESPITE A SHARP DECLINE IN the incidence of gonococcal infection in developed countries during the last decade, gonorrhea remains one of the most common sexually transmitted infections (STIs) in developing countries and a global health problem.1 In the absence of effective vaccine, control of gonococcal infection mainly depends on identification and treatment of the infected individuals and reductions in sexual risk behavior. Early and successful antibiotic treatment of gonococcal infection is important for cure of the patient, prevention of complications, and to reduce transmission.2 Strategies for control of gonorrhea have relied on the use of highly effective and often single-dose therapy administered at the time of diagnosis.3 Information on antimicrobial susceptibility of Neisseria gonorrhoeae is therefore important to guide selection of an appropriate antimicrobial agent.4 Antimicrobial resistance in gonococci often spreads rapidly between countries, and infected travelers often appear for treatment in countries distant from the place of contact.5 Hence, local and regional antimicrobial resistance data are important for management of gonorrhea. Specific data on the incidence of gonorrhea and antimicrobial resistance of N. gonorrhoeae in Nepal is lacking. A pilot study was conducted to assess the effectiveness of current recommendations for treatment for gonorrhea, which is a single oral dose of 500 mg ciprofloxacin. Materials and Methods Bacterial Strain A total of 16 gonococcal isolates isolated from symptomatic and asymptomatic males (n = 65) and females (n = 280) attending an STI service delivery clinic in Eastern Nepal between May and September 2003 were cultured on modified Thayer-Martin medium (MTM). Isolates were presumptively identified as N. gonorrhoeae by Gram stain, oxidase, and superoxol test. Isolates were stored at −86°C at the National Public Health Laboratory, Kathmandu, Nepal, and were transported to ICDDR, B: Centre for Health and Population Research, Dhaka, Bangladesh, for confirmation of the identity by polymerase chain reaction (PCR) and for further analysis.6 Minimum Inhibitory Concentrations Minimum inhibitory concentrations (MICs) of penicillin (Sigma, St. Louis, MO), tetracycline (Sigma), ciprofloxacin (Bayer, Hampshire, U.K.), ceftriaxone (Sigma), spectinomycin (Upjohn, Puurs, Belgium), and azithromycin (Pfizer Inc., CT) for the isolates were determined by the agar dilution method.7,8 The breakpoint criteria defined by the National Committee for Clinical Laboratory Standards (NCCLS) was used for penicillin, tetracycline, ciprofloxacin, ceftriaxone, spectinomycin and the breakpoint criteria used for azithromycin was MIC ≤0.25 μg/mL for susceptible, ≥0.5 μg/mL for reduced susceptible, and ≥1 μg/mL for resistance.7–9 Briefly, 1 × 104 cfu of the bacterial suspension was spotted on GC agar plates (Becton Dickinson, MD) containing Kellogg’s supplement and a 2-fold serial dilution of antimicrobial agents using a multipoint inoculator (Mast Diagnostic Ltd., SCAN 114).6N. gonorrhoeae reference strains WHO A, B, C, D, E, G, H with known MICs were included for quality control in each test. Each test was repeated 3 times. Phenotypic Characterization The criteria used for phenotypic characterization of N. gonorrhoeae were based on plasmid and chromosomally mediated resistance to penicillin and tetracycline as described earlier.10 β-lactamase Test All penicillin-resistant isolates were tested for β-lactamase production by a paper acidimetric method as described earlier.11 Plasmid Typing for PPNG and TRNG Isolates The plasmid type of penicillinase-producing N. gonorrhoeae (PPNG) isolates was (as determined by β-lactamase test) determined by amplification of β-lactamase-producing plasmid as described earlier,12 and the plasmid type for tetracycline-resistant N. gonorrhoeae (TRNG) isolates was determined by amplification of tetM gene in 25.2-MDa conjugative plasmid by PCR as described earlier.13 The primers for PPNG were designed to identify and distinguish Asia- (7426); Africa- (5599) and Toronto- (5154) type plasmid generating a 4.9-kb, 3.1-kb, and 2.6-kb amplicon, respectively (however, the primers did not distinguish between 4.4- and 3.9-MDa PPNG plasmid). The primers for TRNG were designed to identify and distinguish the American- and Dutch-type plasmid-generating amplicon of 1600 bp and 700 bp, respectively. Results A total of 16 gonococcal isolates from males and females with urethral and vaginal discharge symptoms were analyzed. Among the isolates, 2 (12.5%) were resistant (MIC ≥2 μg/mL) to penicillin, including one (6.3%) PPNG isolate; 8 (50%) were resistant to tetracycline (MIC ≥2 μg/mL), including one (6.3%) TRNG; 14 (87.5%) were resistant to ciprofloxacin (MIC ≥1 μg/mL); and 3 (19%) exhibited reduced susceptibility to azithromycin. All isolates were susceptible to ceftriaxone, cefixime, and spectinomycin (Fig. 1). There was no difference in susceptibility of the isolates collected from male and female patients. The distribution of MIC of the antimicrobial agents for the isolates is also shown in Figure 1.Fig. 1: Antimicrobial susceptibility and minimum inhibitory concentration distribution of Neisseria gonorrhoeae strains (n = 16) to (A) penicillin, (B) tetracycline, (C) ciprofloxacin, (D) ceftriaxone, (E) spectinomycin, and (F) azithromycin. Symbols: Susceptible (▧) moderately susceptible or strains with reduced susceptibility () and resistant (▪). The breakpoint criteria used for assessing the susceptibility were as recommended previously.7,8,11The PPNG isolate carried a 3.2-MDa African-type plasmid, and TRNG isolates harbored American-type tetM gene in the conjugative plasmid. Based on plasmid and chromosomally mediated resistance to penicillin and tetracycline, the isolates were phenotypically categorized into 5 different groups (Table 1), and the susceptibility patterns of ciprofloxacin-resistant isolates were further analyzed. Six ciprofloxacin-resistant isolates had chromosomally mediated resistance to tetracycline (Tet R) and one had chromosomally mediated resistance to penicillin (Pen R).TABLE 1: Phenotypic Categories of Neisseria gonorrhoeae Based on Plasmid and Chromosomally Mediated Resistance to Penicillin and Tetracycline (n = 16) and Distribution of Ciprofloxacin Resistance (CipR) Isolates Among Different PhenotypesDiscussion The control of gonococcal infection is important considering the high incidence of acute infections, complications, and sequelae, and its role in facilitating HIV acquisition and transmission.14,15 The knowledge of antimicrobial susceptibility of N. gonorrhoeae is a prerequisite for proper treatment and control of the disease. Regional program for monitoring gonococcal antimicrobial susceptibility have been developed in developed countries such as the United States, Canada, Australia, and The Netherlands; however, in developing countries where the disease burden is high and the resistance is greatest, such activity rarely exists.16N. gonorrhoeae has rapidly developed resistance to most antimicrobial agents and most recently to fluoroquinolones used in the treatment of the disease. Large-scale uses of the drug in an area where over-the-counter availability of the drug without prescription is common may cause a substantial increase in resistance to occur. Although there are some information on the antimicrobial susceptibility of N. gonorrhoeae from Bangladesh and India, data from Nepal is lacking.6 Although more than 80% of the isolates in the current study were resistant to ciprofloxacin, similar resistance patterns have been observed in China.16 Studies on the correlation between in vitro susceptibility and treatment outcome for ciprofloxacin in N. gonorrhoeae has shown that isolates resistant to ciprofloxacin often do not respond to ciprofloxacin therapy.17 Although none of the isolates was resistant to azithromycin, 19% of the isolates showed reduced susceptibility, and treatment failure with such isolates has been reported.9 Data on antimicrobial susceptibility of N. gonorrhoeae in Nepal is lacking and to our knowledge, the limited data from the current pilot study is the first documentation of antimicrobial susceptibility of N. gonorrhoeae in Nepal. National STI case management guidelines in Nepal recommended ciprofloxacin as first-line therapy for the management of uncomplicated gonococcal infection.18 Limited data from the current study demonstrate the need for continuous monitoring of antimicrobial susceptibility of N. gonorrhoeae in Nepal to revise the national STI case management guidelines for treatment of N. gonorrhoeae.

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